Project Summary Nanotechniques involve the creation, characterization, and modification of organized nanomaterials to serve as building blocks for the construction of submicro or nanoscale devices and systems with applications in technology and medicine. Living systems contain a wide variety of ordered macromolecular structures and powerful bionanomachines. The ingenious, novel design of the bacteriophage phi29 DNA packaging motor and its constituents has inspired the artificial synthesis and assembly of phi29 biomimetic motor and its components. The 30-nm nanomotor was geared by six copies of ATP-binding packaging RNAs (pRNAs). The structural versatility of pRNA coupled with its ability to form dimers, trimers, hexamers and patterned superstructures via interlocking loop interactions make it a promising tool for nanomachine fabrication, pathogen detection and drug/gene delivery. The low-resolution global structures of the pRNA and its dimer, trimer and hexamer have been probed by photoaffinity cross linking, chemical modification interference, compensatory modification, Cryo-AFM and 3D computer modeling. The long-term objective is to introduce exogenous components, nano-materials, modules, moieties, drugs, and other therapeutic reagents into the motor and develop it for various applications in nanotechnology and medicine. During the first four years of this grant, we have demonstrated that pRNA can be used as a building block for bottom-up assembly of polyvalent nanoparticles to deliver siRNA, ribozymes and other therapeutics to specific cells. A single molecule fluorescence dual-view imaging system was designed and constructed to count the number of pRNA copies within the constructed pRNA nanoparticles. The short-term objective of this proposal is to utilize prostate cancer as a model system to determine the feasibility of using the phi29 pRNA motor for targeted delivery of siRNA or other therapeutic molecules and treatment of cancerous diseases. Specifically, the novel RNA nanotechnology approach previously developed for the bottom-up assembly of multimeric pRNA nanoparticles will be exploited to construct polyvalent pRNA vehicles carrying an RNA aptamer to specifically bind to prostate surface antigens and a therapeutic siRNA targeting an anti-apoptosis factor for the prostate cancer treatment. Methods for large scale production of pRNA nanoparticles and construction of stable pRNA therapeutic nanoparticles resistant to RNase digestion in vivo will be developed. Approaches designed to combine therapy with the simultaneous detection of the therapeutic effect will be developed by exploiting the polyvalent nature of the pRNA nanoparticles. The efficacy in prostate cancer therapy, the biodistribution, pharmacokinetics, toxicity, off-target effects as well as the double stranded RNA induced interference will be evaluated in both prostate cancer cells and mice models.